Simplified tissue processing

ABSTRACT

Improved systems and methods for tissue processing are described here. The chemical process and the construction of the apparatus are simplified by using only two different solutions in two separate reaction modules. They are compatible with processing of tissue specimens for genetic analysis, histology, in situ antibody binding and hybridization, archival preservation of morphology and nucleic acids, and combinations thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional U.S. Appln. No.60/513,560, filed Oct. 24, 2003.

This application is related to U.S. application Ser. No. 09/735,918,filed Dec. 14, 2000, now U.S. Pat. No. 6,793,890 (which claims prioritybenefit of provisional Appln. No. 60/170,545, filed Dec. 14, 1999);which is a continuation in-part of U.S. application Ser. No. 09/136,292,filed Aug. 19, 1998, now U.S. Pat. No. 6,207,408 (which claims prioritybenefit of provisional Appln. No. 60/056,102, filed Aug. 20, 1997); allof which are incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a chemical process and an apparatus fortissue processing, which are simplified by using only two differentsolutions in two separate reaction modules.

2. Description of the Related Art

Processes for tissue processing have been described which require use ofmore than two different solutions, products useful therein, and systemsfor tissue processing (see WO 99/09390, WO 01/44783, and WO 01/44784).They addressed the need for rapid tissue processing. It is now shownthat (i) a non-aqueous admixture and (ii) a wax solution in two separatereaction modules are sufficient to process tissue quickly andcompletely.

In contrast to the invention disclosed herein, Boon et al. (Eur. J.Morphol. 33:349-358, 1995) use an isopropanol solution and a paraffinwax solution in two separate reaction chambers, each subject to vacuumand microwave heating, to process tissue specimens for histology. Theirsystem requires that tissue specimens be fixed prior to processing anduses a turntable to distribute microwave energy. In addition, the glasscontainer holding the tissue specimens also adsorbs microwave energy andtransfers heat to the solution therein.

Milestone (WO 98/05938) provides another alternative for tissueprocessing in at least three steps: fixing the tissue specimen,simultaneously dehydrating and clearing with dehydrating agent and anessentially lipophilic agent, and impregnating the tissue specimen.Microwave heating is used in the first two steps and elevated pressureis used during the dehydrating/clearing step. The tissue specimen isdried by heating and reduced pressure, and then it is impregnated undervacuum or, alternatively, by applying a cycle of moderate pressure andmoderate vacuum.

The cost of automated instruments for tissue processing and expendituresfor their operation need to be decreased for efficient delivery ofhealthcare. Better diagnosis also raises the confidence of patients thattheir treatment will be based on reliable information and they can befollowed by their physicians in a timely manner. A reduction inequipment costs may allow the purchase of additional instruments so theymay be distributed across the service area (e.g., primary carefacilities). Tissues may be processed locally in such a distributivenetwork and then transported to a specialized pathology facility foranalysis. In physician offices and smaller clinics, space is preciousand decreasing the size of the automated instrument will allow itsplacement in a cramped room.

For example, reducing the number of modules in a tissue processingsystem to two (i.e., one heating module and one vacuum module) willeliminate duplication of mechanical and electrical components and shrinkthe size of the system. It will be more affordable and easier to placein the laboratory. Fewer chemical compositions will need to be stockedand training personnel in use of the system will be eased.

Processes and systems for tissue processing are now described which usefewer chemical compositions in the former and fewer mechanical andelectrical components in the latter as compared to our previouslydescribed inventions. This is an improvement over the processes andsystems disclosed in U.S. Pat. No. 6,207,408 and U.S. Pat. No.6,793,890.

Other advantages of the invention are discussed below or would beapparent to a person skilled in the art from that discussion.

SUMMARY OF THE INVENTION

It is an object of the invention to provide processes and systems fortissue processing which use only two different chemical compositions inthe process and minimizes the number of reaction modules in the system.

The system is comprised of (a) one first module in which the reactioncontents are at least heated, (b) one second module in which thereaction contents are at least under vacuum and heated, and optionally(c) a conveyance which at least transfers one or more tissue specimensat a time from the first module to the second module. Optional loadingand/or unloading stations may also comprise the system.

The first reaction module is comprised of (i) a reaction chamber whichis heated and (ii) a non-aqueous admixture. The non-aqueous admixture iscomprised of different chemicals with at least three differentfunctions: fixing, dehydrating, and clearing. The reaction chamber ofthe first module may be comprised of a microwave source with aprobe/control circuitry to regulate the microwave energy emanating fromthe source. Such energy may be used to heat the contents of the reactionchamber to a preset temperature. The module may be further comprised ofa heated reservoir to preheat the non-aqueous admixture before transferto the reaction chamber and to maintain the temperature of thenon-aqueous admixture after it is transferred from the reaction chamber.The tissue specimen may be substantially hardened by the chemicalcomposition, microwave radiation, or both; and may be initiallyimpregnated by a liquid hydrocarbon (e.g., mineral oil) and thensubstantially impregnated by a wax.

The second reaction module is comprised of (i) a reaction chamber whichis heated and also connected to a vacuum pump and (ii) a wax solution.The reaction chamber of the second module may be comprised of controlcircuitry and appropriate probes to regulate the temperature andpressure therein. The module may be further comprised of a reservoir. Inthe optional reservoir of the second module, solid wax may be meltedtherein and then maintained as a solution until it is transferred to thereaction chamber.

Alternatively, the system is comprised of a single module in which thereaction contents are successively at least heated and then are at leastunder vacuum and heated. One or more tissue specimens remain in the samereaction chamber during processing. A non-aqueous solution and a waxsolution are successively transferred from two separate reservoirs intothe reaction chamber. An optional conveyance may be used to transfer thetissue specimens from optional loading and/or unloading stations.

Further aspects and advantages of the invention will be apparent to aperson skilled in the art from the following detailed description andclaims, and generalizations thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of one embodiment of the invention. A non-aqueousadmixture is stored in a first reservoir 1 and is transferred to amicrowave retort 2 to contact the tissue specimens. Tissue specimens arethen transferred from microwave retort 2 to vacuum retort 3. A waxsolution is stored in a second reservoir 4 and is transferred to avacuum retort 3 to contact the tissue specimens.

FIG. 1B is a schematic of an alternative embodiment of the invention.Tissue specimens are processed in a microwave/vacuum retort 5. Anon-aqueous admixture is stored in a first reservoir 6 and a waxsolution is stored in a second reservoir 7. The non-aqueous admixtureand then the wax solution are transferred to microwave/vacuum retort 5to contact the tissue specimens.

FIG. 2 shows the 18S and 28S rRNA bands after separation by denaturinggel electrophoresis. RNA extracted from fresh mouse liver was used asthe positive control (lane C). RNA was extracted from processed livertissue of two mice (lanes 1 and 2 from the first mouse; lanes 3 and 4from the second mouse). Lanes 1 and 3 were processed for 15 minutes ineach reaction; lanes 2 and 4 were processed for 30 minutes in eachreaction.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

With regard to the processing and analysis of solid tissue, a tissueslice must be on the order of 3 to 6 microns to be examined under amicroscope, whereas the thinnest slice of fresh tissue that can beobtained by cutting is about 1 mm with the typical slice being on theorder of about 2-3 mm. In order to produce a sufficiently thin slice formicroscopic examination, it is necessary to harden the tissue so that athinner slice can be obtained (e.g., by sectioning with a microtome).

In the present invention, the number of (a) different chemicalcompositions and (b) mechanical and electrical components of the systemrequired for tissue processing has been decreased as compared to theprior art. The system is comprised of a single first reaction module inwhich the reaction contents are at least heated (e.g., a heating unit);a single second reaction module in which the reaction contents are atleast under vacuum (e.g., a vacuum unit); and (iii) a conveyance whichat least transfers one or more tissue specimens at a time from the firstmodule to the second module. Each module is comprised of a reactionchamber and a chemical composition which is optionally contained withinthe reaction chamber.

Chemical compositions are typically transferred between a module'sreaction chamber and its optional reservoir which are in fluidcommunication with each other (e.g., tubing or piping, valves, and pumpswith control circuitry to determine the timing, speed, and direction offlow for a chemical composition). One or more tissue specimens may besubstantially hardened and initially impregnated in the first module,and then the substantially hardened tissue specimens are impregnatedwith wax and embedded in a block which is capable of being sectioned.Agitation (e.g., aeration, cycles of vacuum and increased pressure,shaking, etc.) may be used to accelerate exchange between tissuespecimen and chemical composition. Successful completion of tissueprocessing is indicated by the ease and quality of sectioning duringmicrotomy and histologic examination of the sections.

Tissue specimens are hardened with a non-aqueous admixture of chemicals.A suitable admixture is a non-aqueous solution comprised of fixing,dehydrating, and clearing functions (e.g., at least two or threedifferent chemicals). Exemplary agents with fixing and/or dehydratingfunctions are ketones, alcohols, or a combination thereof. For anon-aqueous admixture of fixative and dehydrant, the volume ratio of thetwo agents may be between about 1:10 to about 10:1 (although suchextremes may change the processing time or results may be lessreliable); greater than about 1:6, about 1:3, or about 1:2; less thanabout 2:1, about 3:1, or about 6:1; about 1:1, or any intermediate rangethereof (e.g., between about 1:1 to about 6:1). Clearing function may beprovided by an aliphatic hydrocarbon, benzene, limonene, mineral oil,toluene, and xylenes; mineral oil is a preferred clearant because it isnonflammable and nonvolatile. The tissue specimen may be incubated forat least about 2 minutes, at least about 5 minutes, at least about 10minutes, at least about 15 minutes, at least about 30 minutes, at leastabout 45 minutes, or at least about 60 minutes. The temperature ofincubation may be between about 30° C. and about 80° C.; greater thanabout 40° C., about 50° C., about 55° C., or about 60° C.; less thanabout 70° C. or about 75° C.; or any intermediate range thereof (e.g.,between about 55° C. and 75° C.).

A tissue specimen which has been hardened may then be impregnated with awax solution. Consistent with dehydration of the tissue specimen, thewax solution is preferably as low as possible in water content. Thus,the wax solution may be prepared prior to impregnation by heating thewax to evaporate any dissolved water and by degassing under reducedpressure. Impregnation of the tissue specimen may take place under lessthan atmospheric pressure and at elevated temperature to remove anysolvents from the tissue specimen and to draw the wax solution into thetissue specimen. Vacuum decreases impregnation time by acceleratingdiffusion and reducing the evaporation temperature of any solvents thatmay be present in the specimen. The wax solution may comprise paraffinand/or other waxes, which has been degassed and dehydrated. The tissuespecimen may be incubated for at least about 3 to about 10 minutes, atleast about 15 minutes, at least about 30 minutes, at least about 45minutes, or at least about 60 minutes. The wax solution may be a solidat room temperature (e.g., below about 25° C. or about 30° C.) andmolten above about 55° C. or about 60° C. The temperature of incubationmay be between about 50° C. and about 90° C., greater than about 55° C.or about 60° C.; less than about 75° C., about 80° C., or about 85° C.;or any intermediate range thereof (e.g., between about 55° C. and 85°C.). It is preferred that the incubation be conducted under reducedpressure (e.g., about 100 torr or below about 760 torr). Prior tosectioning, the impregnated tissue specimen may be embedded in theimpregnating agent to form a tissue block.

The reaction chamber may be comprised of any combination of thefollowing: a closure adapted to fit the reaction chamber and to isolatethe reaction chamber from the operator's surroundings (e.g., a lidattached or removable from the reaction chamber); thermal insulation toretain heat in the reaction chamber; at least one temperature and/orpressure probe to monitor conditions in the reaction chamber; a seal toisolate electronic components from chemicals in the reaction chamber;and control circuitry which receives input from at least one probeand/or timer. Similarly, a heated reservoir may be comprised of anycombination of thermal insulation, at least one temperature and/orpressure probe, a seal, and control circuitry which receives input fromat least one probe and/or timer. A rubber gasket is preferred for thereaction chamber (as well as optional heated reservoir) of the firstmodule to isolate fumes of the non-aqueous admixture from the operator.For the second module, a vacuum seal is preferred for the reactionchamber.

In a preferred embodiment, the non-aqueous admixture is premixed andstored in a bottle prior to use. The bottle is opened and at least someof its contents drawn into a first reservoir to be preheated. Solid waxis melted in a second reservoir, and then the wax solution is drawn intothe reaction chamber of the second module. The non-aqueous admixture istransferred between reservoir and reaction chamber of the first moduleduring tissue processing; in contrast, the wax solution is maintained inthe reaction chamber of the second module during tissue processing. Atthe end of the day, the non-aqueous admixture is drawn back into thebottle, the wax solution is drawn back into the second reservoir, andthe solutions are safely disposed or stored for reuse.

Alternatively, a single reaction chamber may be used within a modulewhich combines both heating and vacuum functions in a single unit. Insuccession, the non-aqueous admixture and the wax solution aretransferred from separate first and second reservoirs, respectively, tothe reaction chamber and back again.

Such system may be manually operated or automated (i.e., transfer oftissue specimen between modules with a mechanical conveyance). Anautomated system may be further comprised of a loading and/or unloadingstation. Tissue specimens may be loaded into the system and processedeither in batches or as separate specimens. Tissue specimens may enterthe system at the loading station and exit the system from the unloadingstation where they are optionally collected. Control circuitry (e.g.,hardware and software) may be used to program the movement of tissuespecimen(s) in the system, to prevent access to the reaction modulesduring operation, to modify reaction parameters (e.g., time,temperature, pressure, or amounts of chemicals of the process), or anycombination thereof.

Here, a “tissue specimen” is a piece of tissue that may be processed bythe methods disclosed herein. It may also refer to single cells from anybiological fluid (e.g., ascites, blood, pleural exudate), or cellsuspensions obtained from aspiration of solid organs or lavage of bodycavities. Single cells may be pelleted by buoyant centrifugation orsedimentation prior to processing. Solid pieces (i.e., tissue slices)are commonly processed for histology and pathology.

Fixing function may be provided by ketones (e.g., acetone, methyl ethylketone); aldehydes (e.g., acetylaldehyde, formaldehyde, glutaraidehyde,glyoxal); low-molecular weight alcohols (e.g., methanol, isopropanol,ethanol, propanol, butanol, isobutanol, ethyl butanol, amyl alcohol); orthe like. Fixative in the non-aqueous admixture may be greater thanabout 15% (v/v), 20% (v/v), about 25% (v/v), about 30% (v/v), about 35%(v/v), or about 40% (v/v); less than about 35% (v/v), about 40% (v/v),about 45% (v/v), about 50% (v/v), about 55% (v/v), about 60% (v/v), orabout 65% (v/v); or any intermediate range thereof (e.g., between about20% and 60%). Dehydrating function may be provided by low-molecularweight alcohols (e.g., methanol, isopropanol, ethanol, propanol,butanol, isobutanol, ethyl butanol, amyl alcohol); ketones (e.g.,acetone, methyl ethyl ketone); or the like. Dehydrant in the non-aqueousadmixture may be greater than about 15% (v/v), 20% (v/v), about 25%(v/v), about 30% (v/v), about 35% (v/v), or about 40% (v/v); less thanabout 35% (v/v), about 40% (v/v), about 45% (v/v), about 50% (v/v),about 55% (v/v), about 60% (v/v), or about 65% (v/v); or anyintermediate range thereof (e.g., between about 20% and 60%). Incontrast to conventional fixation with aldehydes (e.g., formalin), useof ketones and alcohols is believed to act as fixatives by physicallystabilizing proteins (e.g., precipitation) without chemically combiningwith them. Aliphatic hydrocarbons, benzene, limonene, mineral oil,toluene, and xylenes may be used for their clearing function; mineraloil is preferred. Clearant may be greater than about 10% or about 15%(v/v) of the non-aqueous admixture; it may be less than about 25% orabout 30% (v/v) of the non-aqueous admixture. Processing may beaccelerated by addition of a surfactant: e.g., dimethyl sulfoxide(DMSO), polyoxyethylene sorbitan esters (e.g., TWEEN 80), sodiumdimethyl sulfosuccinate, mild household detergents, or the like. Thenon-aqueous admixture may also be buffered with the appropriate use ofacid and base.

Microwave radiation may also assist hardening of the tissue in aphysical rather than chemical manner. The combination of physical andchemical processes may decrease processing time and/or increase specimenquality. The effect of a particular physical or chemical treatment canbe determined by noting the effect on tissue processing of omitting thetreatment.

Finally, the tissue specimen is impregnated with an agent such as wax(e.g., paraffin), mineral oil, or non-water-soluble waxes. Preferredimpregnating agents are commercial wax formulae, mixtures of waxes ofdifferent melting points (waxes are solid at room temperature and havemelting points which are dependent on their chain lengths, while mineraloil is liquid at room temperature), and the like.

The tissue specimen may be fresh, partially fixed (e.g., fixation in 10%formalin for 2-3 hours), or fixed (e.g., overnight fixation in 10%formalin or any other fixative). The above process allows processing ofa tissue specimen from fixation to impregnation in less than about twohours, less than about 90 minutes, less than about 60 minutes, less thanabout 30 minutes, or less than about 15 minutes. The time required forsolution in each step to reach the appropriate temperature isinsignificant compared to incubation time for each step, and may bedisregarded to calculate the total time for processing. In particular,small biopsies and tissues less than about 1.5 mm thick, as well asthose containing little or no fat, could be processed quickly. Tissuemay be transported from the operating room to the pathology laboratoryin a non-aqueous admixture as disclosed in published U.S. Appln.2003/0211452: UM-FIX as used here is about 10% low molecular weight,polyethylene glycol (PEG) and about 90% methanol. This preservative wasalso characterized in Vincek et al. (Lab. Invest. 83:1-9, 2003). Withthe exception of PEG that is present from tissue preserved in UM-FIX andwhich is carried into the process of the present invention, PEG is notused and does not appear to be required for tissue processing. Thissimplifies the chemistry of the present invention and reduces the numberof incubations which are necessary in comparison to the previousprocess.

Following impregnation, the tissue specimen can be embedded to produce ablock. The agent used to embed the tissue specimen is preferably thesame as the material used for impregnation, but a different impregnatingagent may also be used. The blocked tissue specimen can be mounted on amicrotome to produce tissue sections of between about 1 micron and about50 microns, or between about 2 microns and about 10 microns. Tissuesections may be further processed for histochemical staining, antibodybinding, in situ nucleic acid hybridization or amplification, or acombination thereof. The tissue specimens are then typically examined bymicroscopy, but other techniques for detecting cellular properties maybe used to examine the processed tissue specimen (e.g., automatedcytometry, autoradiography, electrophoresis of nucleic acid). Tissueblocks may be stored for archival purposes or retrospective studies.

Cell phenotypes (e.g., reactivity with cell-specific antibody, chemicalstaining) may be analyzed by removing the embedding material (e.g.,deparaffinization) and dissecting tissues (e.g., proteolytic digestionand mechanical disaggregation). Single cells may be dispersed in a sprayand analyzed in a flow cytometer. Alternatively, deparaffinized tissuemay be mounted on a microscope stage, dissected with a laser and/ormicromanipulator into substantially homogeneous cell populations, andthe different cell types analyzed by physical, chemical, or genetictechniques.

The present invention is compatible with preparation of nucleic acids,DNA or RNA, from processed tissues. Thus, genetic study is possible forspecimens collected routinely in the clinical pathology laboratory. Thecombined power of these technologies will be great, Histologicalobservations may be correlated with genetics by analyzing one section bystaining or immunohistochemistry, and preparing nucleic acids from anadjacent section for genetic analysis. For example, diseased and normalregions of the same section may be compared to detect geneticdifferences (e.g., mutations, levels of transcription), diseaseprogression may be characterized by comparing genetics differences insamples taken at several time points, and tumor evolution may beassessed by following the accumulation of genetic differences fromprimary cancer to metastasis. Substantially homogeneous or merelyenriched cells may be obtained by sorting with a flow cytometer ormicrodissection.

Mutations may be germline and used to trace genetic predisposition ofdisease, or mutations may be somatic and used to determine geneticalterations in disease pathogenesis. The disease may be a metabolic orneurologic disorder, malignancy, developmental defect, or caused by aninfectious agent. The present invention preserves material for geneticanalysis by a simple procedure and room temperature storage. It may beanalyzed by in situ hybridization or nucleic acids may be extracted fromtissue.

Hematoxylin-eosin staining is commonly used for histological study andmay be considered a standard for comparison by pathologists. Inaddition, the present invention may be compatible with other stainsincluding trichrome, reticulin, mucicarmine, and elastic stains asdescribed in general references such as Thompson (Selected Histochemicaland Histopathological Methods, C. C. Thomas, Springfield, Ill., 1966),Sheehan and Hrapchak (Theory and Practice of Histotechnology, C. V.Mosby, St. Louis, Mo., 1973), and Bancroft and Stevens (Theory andPractice of Histological Techniques, Churchill Livingstone, New York,N.Y., 1982). Such staining procedures would take between 30 minutes andseveral hours to complete, although rapid staining procedures areavailable from Fisher Scientific that require only five minutes toaccomplish.

Tissue may be obtained from an autopsy, a biopsy (e.g., endoscopicbiopsy), or from surgery. Small specimens such as punch or needlebiopsies obtained with a trochar are well suited for the presentinvention. For cancer surgery, the ability to provide a pathologicaldiagnosis from a stained tissue section will provide the surgeon withinformation that may be used prior to the patient's departure from theoperating room. For example, an indication from the pathologist that thecancer is confined to the resected tissue may allow the surgeon to beconservative in treatment and to preserve neighboring healthy tissue.Alternatively, a finding by the pathologist that cancer is not confinedto a resected organ would permit more aggressive surgical treatmentwhile the patient was still in the operating room. In contrast toconventional histological processing of frozen tissue, the processing offresh tissue in accordance with the invention may provide tissuespecimens with better morphology and reduce the need for laterconfirmation by a pathologist viewing fixed tissue.

Exemplary tissues which may be processed in accordance with theinvention include: appendix, bladder, bone, bowel, brain, breast,carcinoma, cervix (squamous epithelium), gall bladder, heart, kidney,liver, lung, ovary, parotid gland, placenta, prostate, skin, spleen,testicle, thyroid gland, tonsil, and uterus (myometrium andendometrium). Lymphoreticular and fatty tissues may be processed inaccordance with the invention. Mineralized tissue would requiredecalcification prior to processing by the present process. Subsequentanalysis would include detecting DNA mutations and RNA expression,genomics, histology, immunochemistry, and proteomics.

Tissue sections processed by the present process may also be used inimmunohistochemistry. The present process provides tissue specimens inwhich antigen is recovered and preserved, the choice of fixative may beoptimized for recovery and preservation of particular antigens.Non-specific binding sites are blocked, antigen is bound by specificantibody (i.e., the primary antibody), and non-bound antibody isremoved. If labeled with a probe or signal generating moiety, theprimary antibody may be detected directly but it is preferred to attachthe probe to a protein (e.g., a secondary antibody) that specificallybinds the primary antibody. Secondary antibody may be raised against theheavy or light chain constant region of the primary antibody. Thisamplifies the signal generated by an antigen-antibody conjugate becauseeach primary antibody will bind many secondary antibodies.Alternatively, amplification may occur through other specificinteractions such as biotin-streptavidin. Antibody binding is performedin a small volume to reduce usage of expensive reagents and maintain ahigh binding rate; evaporation of this small volume is reduced byincubation in a humidity chamber. The signal generating moiety ispreferably an enzyme which is not otherwise present in the tissue. Forexample, alkaline phosphatase and horseradish peroxidase may be attachedto the secondary antibody or conjugated to streptavidin. Substrates areavailable for these enzymes that generate a chromogenic, fluorescent, orluminescent product that can be detected visually.

The staining pattern for antigen may be used to localize expression ofthe antigen in the context of cellular structures revealed bycounterstaining. Antigen expression can identify cell or tissue type,developmental stage, tumor prognostic markers, degenerative metabolicprocesses, or infection by a pathogen.

Antigen-antibody binding may also be visualized with fluorescent,radioactive, or colloidal metal probes by epifluorescence,autoradiography, or electron microscopy. Similar probes may be used todetect nucleic acid in the tissue section by in situ hybridization toidentify genetic mutations or transcripts; alternatively, the nucleicacid (DNA or RNA) may be extracted from tissue sections and analyzeddirectly by blotting, or amplified prior to further genetic analysis.

Tissue processing may be integrated with other automated systems: e.g.,an embedding and sectioning system which produces a block ofparaffin-embedded tissue; a microtomy, staining, and coverslippingsystem which provides slides containing tissue sections; a developingsystem which visualizes histochemical and/or immunochemical signals onthe tissue sections; a flow cytometer which analyzes and/or sorts singlecells by their phenotypes into substantially homogeneous or merelyenriched populations; a microdissection system which separates tissuesinto substantially homogeneous populations; an imaging system whichscans tissue sections on a slide, digitizes signals visualized through amicroscope, and then manipulates, stores, and transmits those images;and combinations thereof. Cells or tissues, especially those sortedand/or separated into substantially homogeneous populations, may befurther analyzed by their DNA or RNA sequences, genetic mutations,changes in the level or pattern of gene expression, changes in the levelor pattern of protein expression, and combinations thereof. Systemintegration and data management is facilitated by identifying tissuespecimens or their holders by alphanumeric characters, bar code,radiofrequency identification (RFID), or other labeling. The same ordifferent labels may be used to identify a particular tissue specimen asit proceeds through a series of automated systems. The labels and otherinformation about the specimen (e.g., patient name, date, location inthe facility, disease or other pathological condition, tissue type,diagnosis, phenotype, genotype, genomic or proteomic characterization)may be entered into a database management system to store, manipulate,and retrieve the data. Mining such information in the database may proveor disprove correlations in accordance with statistical criteria, andsuggest further investigations.

An initial step in the process, which may be carried out in the surgicaltheater, pathology laboratory, or elsewhere, is to prepare a suitabletissue specimen for hardening and, ultimately, examination. Typically, aslice of the tissue of interest is prepared. A fine slice or trocharsample may be obtained for processing: about 0.5 mm to about 1 mm thick,about 1 mm to about 3 mm thick, about 1 mm to about 2.5 mm thick, orabout 1.5 mm to about 2 mm thick. The tissue slice is placed in a tissuecassette or other holder in which the tissue is contained duringsubsequent processing until the hardened specimen is ready forsectioning. Alternatively, a tissue specimen is prepared by coring orsnipping tissue with a trochar (e.g., for biopsy). For ease of handlingmany cassettes, the cassettes may be placed in a carrier or basket. Thecassette or holder is next placed in a non-aqueous admixture inaccordance with the present invention.

A tissue cassette or holder is exposed to the non-aqueous admixturewhich hardens tissue while simultaneously being agitated and subjectedto microwave radiation. A single microwave unit is needed because onlyone hardening solution is needed. The hardening solution may remain inthe reaction chamber through several cycles of tissue processing or itmay be transferred between the reaction chamber and a storage chamber atintervals (e.g., removed to storage chamber after every cycle ofhardening, when all tissue specimens have been processed, or at the endof operations during the day). A carrier of tissue specimens orcassettes containing a solid tissue specimen may be transferred betweenreaction chambers manually, or by an armature or track conveyance.

To provide for agitation which accelerates tissue processing, aerationis provided. A tube may be inserted directly into the non-aqueousadmixture below the tissue specimens, but for more uniform and completeagitation, a diffusion plate at the bottom of the reaction chamber andacross a substantial portion of its diameter may be used for uniformagitation of the entire volume of solution. Agitation may also beprovided by pressure and vacuum (P/V) cycles (e.g., periods of about 10to 30 seconds spent under pressure, reduced to a partial vacuum, andunder pressure again) or pumping solution into and out of the receptacle(e.g., circulating the solution through the receptacle) or using P/Vcycles.

The tissue cassette or holder is placed in a wax solution contained in avacuum unit. Conventionally, wax is degassed as a part of the tissueprocessing procedure. Degassing removes organic solvents and excessmoisture from the wax. To enhance this process and to reuse the wax inthe system, continuous degassing under vacuum may be performed.

Next, the tissue specimen is embedded (preferably with an automatedembedder). The embedded tissue specimen is then sectioned with amicrotome and floated on water for placement on a glass microscope slide(preferably with an automated sectioner). After the section is placed onthe slide, the slide is heated to melt the paraffin and adhere thesections to the glass. The slides are then stained (preferably with anautomated stainer) and covered (preferably with an automatedcoverslipper).

In a preferred embodiment, the system for hardening and impregnating atissue specimen in accordance with the present invention can be limitedto two discrete modules: a microwave unit and a vacuum unit. The tissuespecimen is contacted with the non-aqueous admixture which hardens fixedor unfixed tissue in the microwave unit. Impregnation of the hardenedtissue specimen is finished in the vacuum unit. Only one vacuum unit isrequired for impregnation. Agitation may be provided with a mechanicaldevice that causes aeration in, shaking or vibration of, or transfer ofultrasound energy into the solution. Alternatively, a pump may be usedfor agitation using P/V cycles or circulating the solution.

A microwave unit of the invention is comprised of (i) a source of themicrowave energy (e.g., magnetron, klystron, traveling wave tube), (ii)a waveguide that transmits the microwave energy from the source to areaction chamber, its dimensions and shape being adapted for thispurpose, and (iii) a reaction chamber that receives the transmittedmicrowave energy and is adapted to process a tissue specimen by at leastchemical fixation, dehydration, and defatting. The reaction chamber maycontain a plurality of different tissue specimens. Preferably, theinterior geometry of the reaction chamber is configured to achieveuniform distribution of microwave energy and heating of its contents.Uniformity is achieved primarily by consideration of two factors.

First, the circumference of the reaction chamber is made to be anintegral number of half wavelengths of the microwave radiation in thechamber. With proper arrangement of the waveguide entrance into thereaction chamber, a mode will be excited that will propagate around theexterior wall. This type of mode is characterized by the microwave fieldbeing predominantly near the exterior wall. A similar phenomenon occursin acoustics where sound waves travel very efficiently next to solidwalls. These types of modes are referred to as whispering gallery modes.

A second consideration is the radial distance between the boundary ofsolution in the reaction chamber and its wall. The optimum spacing isdetermined empirically by changing that spacing. If the spacing is toonarrow, the microwave radiation is absorbed primarily near the waveguideentrance to the reaction chamber. If the spacing is too wide, thereaction chamber becomes a resonant cavity and is sensitive to theamount of non-aqueous admixture and solids (e.g., tissue specimens,cassettes, and basket) therein. With the proper spacing, efficientheating of the solution and solids is achieved over an extensive rangeof heights of the contents as measured by a level sensor outside thereaction chamber (i.e., volumes therein). As little as 10% of the fullheight (i.e., total volume) still provides efficient heating of thecontents.

Similarly, the source and the waveguide are configured to achieveminimal energy loss during transmission of the microwave radiation. Themicrowave unit is configured with a waveguide to have no more than about2% energy loss from the source to the reaction chamber. A higher energyloss would require the use of expensive shielding and other protectiondevices for the source of the microwave energy.

Heating may be controlled by cycling power on-off in cycles of about 10to 25 seconds because a minimum time is required by the heatingcharacteristics of the cathode of the microwave source. But this mayburn the tissue, so heating may be controlled through a variable currentsource to allow continuous variation in the power delivered by themicrowave source to the reaction chamber. Such burning or over cookingis typified by homogeneous staining of tissue structures withoutdistinguishing cellular features. The latter is preferred to reduce peakpower output.

The microwave unit may be further comprised of any combination of acontainer adapted to fit in the reaction chamber and to receive at leastone tissue specimen (e.g., a basket); at least one temperature and/orpressure probe to monitor conditions in the reaction chamber; one ormore energy probes to monitor microwave energy being sent by the source,transmitted through the waveguide, and/or received by the reactionchamber; a closure adapted to fit the reaction chamber and to isolatethe reaction chamber from the operator's surroundings; thermalinsulation to retain heat in the reaction chamber; shielding to isolateelectronic components from chemicals in the reaction chamber; andcontrol circuitry to receive input from at least one probe or timer andthereby regulate at least one of the microwave energy from the source,transmitted through the waveguide, and/or received in the reactionchamber. The container is preferably transparent to microwave radiationand therefore energy is not consumed in its heating.

Characteristics of the materials used for the vacuum seal are theability to hermetically isolate reaction chamber or reservoir from theenvironment, optionally substantial transparency to microwave radiation,malleability to ensure a tight fit which conforms to the closure, andchemical resistance to solutions of the process. Modifying a reactionchamber or reservoir with (i) a closure and a gasket/seal to reduceevaporation and (ii) thermal insulation can reduce the power required tooperate the heater or vacuum unit by two- or three-fold.

The modules may occupy the same space and/or the tissue specimen mayremain stationary. Microwave or thermal energy may be regulated andtransmitted into the same space, or onto the stationary tissue specimenat different times in the process. Chemical solutions and/or vapors maybe moved into or out of the same space, or brought into or out ofcontact with the stationary tissue specimen. Preferred is minimizingspace requirements for the system by using two reaction chambers, andtransporting the different chemical compositions into a reaction chamberby tubing or piping from separate storage and/or waste chambers. Acontroller can receive input from the reaction chamber and/or fromtiming that part of the processing cycle, and thereby regulate thetransport of the different chemical compositions.

Either transferring different solutions into and out of the reactionchamber or transferring the basket among reaction chambers containingdifferent solutions may effect changes in reaction steps. Holding thebasket above the interior of the reaction chamber for about 5 to 10seconds allows excess solution to drain back through one or moreopenings in the bottom and/or sides before the basket is transferred.Thus, the sequence in which the basket is transferred among reactionchambers, each containing a particular composition of tissue processingchemicals, and the time the basket is incubated in each reaction chamberwill dictate the series of chemical reactions necessary to accomplishthe process according to the invention.

The lid can be removed; the gasket can be attached to the lid and movedwith it. This process of removing the lid and gasket is performed forboth the reaction chamber which initially contains the tissue specimensand the next reaction chamber into which the tissue specimens will besubsequently transferred. The basket is then removed, allows solution todrain from the basket and any cassettes which may be contained thereinback into the reaction chamber for about 5 to 10 seconds, and transfersthe basket to the reaction chamber containing the next chemical solutionin the process. Flushing of the tubing/piping and cleaning of thereaction chamber are not required because the amount of solution leftbehind is minimal. Finally, the lids and gaskets are replaced. The totaltime for such a transfer is about one minute.

In accordance with the invention, variations on the above embodimentsare envisioned. Various configurations of the tissue processing systemare possible, and optional modules may be connected to form a portion ofthe system. The specific configuration chosen may be dictated by theaverage number of specimens that will be processed on a daily basis bythe clinical laboratory, and/or the speed with which histology orpathology reports must be prepared.

The system may be manually operated or automated. Manual operation isparticularly suited for research and development because variations inthe process or apparatus may be quickly assessed, or in facilitiesprocessing a small number of specimens. For automated instruments,tissue specimens may be transported by a mechanical conveyance (e.g.,robot arm, track formed by belt and roller) and/or chemical compositionsmay be transferred by corrosion-resistant plumbing. Thus, tissueprocessing may be automated by transferring specimens between stationarymodules in a particular sequence, filling and emptying modules ofdifferent chemicals such that stationary tissue specimens are incubatedin a particular sequence, or any combination thereof. Programs whichcontrol parameters of tissue processing (e.g., startup and shutdown ofinstrument, loading and unloading a number of specimens, conditions suchas reaction time, progress of specimens through the system) may bemonitored on a screen; parameters of tissue processing (e.g., specimenin reaction chamber for about 15, 30 or 45 minutes) may be preset orselected by the operator through a keypad.

The armature conveyance may, for example, grab the specimen with apincer-like mechanism or catch the specimen with a hook-like device. Thearm may be articulated to perform human-like motion; or may be mountedin a fixed coordinate rack with linear or two dimensional movement, andoptionally another dimension of movement provided by varying the heightof the arm over the system. The track conveyance may be made fromresilient or tacky material to fix the specimen on the track byfriction, or there may be a regular series of bumps or walls to trap thespecimen therebetween. The track may be formed as a continuous belt ormay be a series of belts that convey the tissue specimen, with the beltput into motion with a roller or sprocket mechanism. The cassette orholder may be adapted for conveyance by having a stem (with or without aknob) to be grabbed or a loop to be caught by the arm, or by fittingwithin a groove or indentation in the track. Similarly, the cassette orholder may be organized in a carrier or basket for processing a largenumber of specimens, the carrier or basket being adapted for transportby the armature or track conveyance.

Electric motors and controllers may be used to transport a tissuespecimen by the operator's real-time command or selection of a storedprogram. A simple mechanism of controlling the time spent by the tissuespecimen in each module would be to move the tissue sample or holderthereof at a constant speed and to adjust the length of the path througheach module to accommodate the intended incubation time.

The flexible tubing or fixed piping, as well as other components of theplumbing, should be made of chemical-resistant materials to preventcorrosion (e.g., glass, stainless steel, polyethylene,polytetrafluoroethylene, polyvinylchloride). Controllers andpumps/valves may be used to transport chemical compositions from storagechamber to reaction chamber, from reaction chamber to storage chamber ifthe composition can be reused, and from reaction chamber to wastechamber if the composition is to be flushed from the system; to fill thestorage chamber; and to flush the waste chamber by the operator'sreal-time command or selection of a stored program. A heated reservoirand heated plumbing components may be necessary to maintain the chemicalcomposition at reaction temperature or to ensure that the chemicalcomposition (e.g., wax solution) is kept in a transportable fluid state.Vapor seals and/or cooling may be necessary to isolate corrosive vaporsfrom the mechanical and electrical components of the system.

All of the patents, patent applications, and other publications cited inthis specification are incorporated herein by reference in theirentirety.

The following examples are meant to be illustrative of the presentinvention, but the practice of the invention is not limited orrestricted in any way by them.

EXAMPLES Example 1

Tissue processing with a series of non-aqueous solutions is described inU.S. Pat. No. 6,207,408. In particular, a series of four solutions wasused to successfully process tissue for histological examination in PCTpublication WO 01/447683 and published U.S. Appln. 2001/0051365.Experience using this prior invention has been described by Morales etal. (Arch. Pathol. Lab. Med. 126:583-590, 2002; Am. J. Clin. Pathol.121:528-536, 2004). Here, we modified the program used in a rapid tissueprocessor with a series of four solutions in separate modules todetermine the contribution of each solution to successful microtomy andhistology. Using these modifications, different solutions in the fourmodules of the system were selected and the results are summarized inTable 1. The requirement for each solution was assessed with a varietyof formalin-fixed tissues which were about 1.5 mm thick.

TABLE 1 Only two solutions are required for successful tissue processingMicrowave Unit Vacuum Unit I II III IV Microtomy Histology 15 min 15 min15 min 15 min +++ +++ 30 min 15 min 15 min + + 45 min 15 min 15 min + ++45 min 15 min 15 min ++ +++ 45 min 45 min − − 45 min 45 min ++ ++ 60 min60 min +++ +++ Solution I: 40% isopropanol, 40% acetone, 20%polyethylene glycol (MW 300), glacial acetic acid added to aconcentration of about 0.5% total volume, and dimethyl sulfoxide (DMSO)added to a concentration of about 1% total volume at about 62° C.Solution II: 55% isopropanol, 25% acetone, 20% mineral oil, glacialacetic acid added to a concentration of about 0.5% total volume, anddimethyl sulfoxide (DMSO) added to a concentration of about 1% totalvolume at about 62° C. Solution III: 30% mineral oil and 70% moltenparaffin at about 65° C. and a pressure of about 640 torr. Solution IV:molten paraffin at about 65° C. and a pressure of about 640 torr.

In a modification of the tissue processing described in Example 4 of WO01/447683 and published U.S. Appln. 2001/0051365, specimens wereprocessed in a tissue processor under variable control. The initialprocess used four modules (i.e., two microwave units and two vacuumunits with four different solutions each) and incubation times of 15minutes per module. Programming the tissue processor to extendincubation times and to skip modules using solutions I and IIIdemonstrated that both the four solution/one hour process and the twosolution/two hour process produced excellent results (+++). To beconsidered excellent, microtomy of tissue blocks had to produceacceptable ribbons of serial sections which could be floated in a waterbath without “exploding” and then positioned on a glass slide forhistological staining without “cracking” consistently and uniformlythrough a variety of tissue types. This handling characteristic oftissue sections after cutting with a microtome was essential to provideefficient and reliable pathological diagnoses based upon the morphologyobserved in stained sections. Portions of the section which were missingor poorly stained would prevent rendering an opinion on the morphologyof the tissue in that section and thereby reduce confidence indiagnostic conclusions. Therefore, procedures and instruments which areused in a clinical pathology laboratory require validation that they canbe consistently and efficiently performed, a variety of tissue types canbe processed and different diagnostic criteria can be applied toprocessed tissues using a standardized protocol, and precious specimenswill be reliably processed. Both processes produce excellent results.But the present invention has the advantage of requiring only twodifferent chemical compositions and half the number of system componentsrequired for tissue processing using a series of non-aqueous solutions.The longer processing time is an acceptable compromise and may bemitigated by use of thinner tissue specimens and/or preprocessing ofspecimens.

During microtomy, poorly processed tissue specimens do not form a ribbonof serial sections, the section explodes when floated in a water bath,and there are cracks (i.e., missing portion) in the section. “Good” and“excellent” tissue processing results do not suffer from such defectsbecause microtomy produces sections with preserved morphology (e.g.,cellular structure and tissue organization) during subsequent analysis(e.g., histological staining, antigen binding by antibody, in situhybridization). But good (++) results are variable: (a) inconsistentwith sections from the same tissue type or (b) consistent with sometissue types but unsatisfactory with other tissue types. Such variableresults are not acceptable for diagnostic purposes because it ispreferable to use the same protocol for all specimens processed in thelaboratory.

Elimination of solution II did not produce excellent results even whenthe time of incubation for solution I was lengthened to 45 minutes.Microtomy showed that paraffin sections floated on a water bath wouldunacceptable spread out on the surface and “explode” prior to placementon glass slides. Histology, however, showed that there were fewer“cracks” after hematoxylin-eosin staining of deparaffinized sectionswhen incubation in solution I was lengthened from 30 minutes to 45minutes.

Therefore, the remaining investigations were performed with solution IIand without solution I. Incubation in solution II was 45 minutes or 60minutes. Processing with 15 minutes each in solutions III and IV did notproduce sections with acceptable handling characteristics. Serialsections did not produce acceptable ribbons although the histology wasadequate. Use of only solution III was disastrous (cf. only solution IIIfor 45 minutes to only solution IV for 45 minutes) because the blockscould not be sectioned. The combination of only solutions II and IVproduced at least good results in regards to microtomy and histology.But incubation times of 60 minutes instead of 45 minutes are preferredbecause the former could process both fresh and formalin-fixed specimenswhile the latter did not acceptably process fresh tissue. The shorterincubation time also gave inconsistent results with “cracks” inlymphoreticular tissues (e.g., spleen) and unacceptable morphology withfatty tissues (e.g., breast). Diverse tissue types have beensuccessfully processed with the longer incubation time (see below).

Example 2

Fresh, formalin-fixed, and UM-FIX-treated tissue specimens of about 1.0mm to about 2.0 mm (preferably about 1.5 mm) in the thinnest dimensionhave been processed, Bladder, breast, carcinoma of the lung, cervix(squamous epithelium), kidney, liver, ovary, spleen, tonsil, and uterus(endometrium and myometrium) gave uniformly excellent results formicrotomy and histology (hematoxylin-eosin staining of deparaffinizedsections).

The non-aqueous admixture is comprised of:

-   -   about 25% acetone    -   about 55% isopropanol    -   about 20% mineral oil    -   glacial acetic acid added to a concentration of about 0.5% total        volume    -   DMSO added to a concentration of about 1% total volume.        The three major components were mixed in a volume of 3.8 L, and        then the acetic acid and DMSO were added. Specimens were        incubated for about 60 minutes in this non-aqueous admixture at        about 62° C.; chemical exchange was promoted by agitating the        non-aqueous admixture (e.g., aeration). Both the specimen and        the non-aqueous admixture were heated by microwave energy during        incubation.

Following hardening of the tissue specimen by chemicals in thenon-aqueous admixture and/or microwave irradiation, the tissue specimenwas impregnated for 60 minutes in molten paraffin, which was degassedand dehydrated, at about 65° C. and a reduced pressure of about 640 mmof Hg. Impregnation was promoted by agitating the wax solution with P/Vcycles. After a total processing time of about two hours, the tissuespecimen was ready for microtomy and subsequent analysis, or storage.

For thicker tissue specimens (e.g., about 2.5 mm thick slice), theincubation times with non-aqueous admixture and wax solution may have tobe lengthened to about 90 minutes or 120 minutes for each solution.

Example 3

Fresh, formalin-fixed, and UM-FIX-treated tissue specimens of about 1.0mm to about 2.0 mm (preferably about 1.5 mm) in the thinnest dimensionhave been processed. Uniformly excellent results for microtomy andhistology (hematoxylin-eosin staining of deparaffinized sections) havebeen obtained for human tissues (e.g., adenoid, breast, kidney, lipoma,liver, placenta, skin, spleen, and uterus) under the followingconditions (i.e., two reactions each for about 30 minutes).

The non-aqueous admixture is comprised of:

-   -   about 50% acetone    -   about 30% isopropanol    -   about 20% mineral oil    -   glacial acetic acid added to a concentration of about 0.5% total        volume    -   DMSO added to a concentration of about 1% total volume.        The three major components were mixed in a volume of 3.8 L, and        then the acetic acid and DMSO were added. Specimens were        incubated for about 30 minutes in this non-aqueous admixture at        about 62° C.; chemical exchange was promoted by agitating the        non-aqueous admixture (e.g., aeration). Both the specimen and        the non-aqueous admixture were heated by microwave energy during        incubation.

Following hardening of the tissue specimen by chemicals in thenon-aqueous admixture and/or microwave irradiation, the tissue specimenwas impregnated for 30 minutes in molten paraffin, which was degassedand dehydrated, at about 65° C. and a reduced pressure of about 640 mmof Hg. Impregnation was promoted by agitating the wax solution with P/Vcycles. After a total processing time of about two hours, the tissuespecimen was ready for microtomy and subsequent analysis, or storage.

For smaller biopsy specimens obtained with a trochar (e.g., a cylinderof about 2 mm to about 3 mm in diameter), the incubation times withnon-aqueous admixture and wax solution may have to be shortened to about15 minutes for each solution.

In other embodiments, appendix, bowel, breast, fallopian tube, kidney,liver, lung, parotid, placenta, prostate, thyroid, and uterus have beenprocessed for microtomy and histology when each reaction time wasshortened to about 20 minutes. In yet other embodiments, adenoma,appendix, breast, cervix, gall bladder, kidney, liver, lung, ovary,skin, spleen, thyroid, tonsil, and uterine body (as well as fresh mouseliver) have been processed for microtomy and histology when eachreaction time was shortened to about 15 minutes.

Example 4

Tissue processing may be performed using an embodiment of the invention(shown in FIG. 1A) in the following manner. A bottle of a non-aqueousadmixture (3.8 L) is attached and paraffin pellets (3 L) are added. Thenon-aqueous admixture is prewarmed and the solid paraffin, which hadbeen degassed and dehydrated when in liquid form, is melted prior toloading of samples. Vacuum is drawn and air pressure is raised totransfer solutions and, if needed, provide agitation of solutions withinthe retort by bubbling (pump BP) or P/V cycling (pump AP). A connection(e.g., flexible tubing) and a port where the connection joins differentcomponents of the system may be used to transfer solution between afirst reservoir and microwave retort using a pump LP and solenoidvalves. Only impregnation in the vacuum retort requires a reduction inthe pressure with an air pump because tissue processing in the microwaveretort (e.g., hardening and initial impregnation) is performed atatmospheric pressure with mechanical agitation using a bubbling pump.

Solutions and retorts are warmed to appropriate operating temperatures.For example, the non-aqueous admixture is preheated to about 55° C. byan electric heater in the reservoir of the first module (first reservoir1) prior to transfer into the microwave retort 2. The non-aqueousadmixture is typically transferred back to the first reservoir 1 priorto opening the microwave retort 2 so that tissue specimens are placed inan empty reaction chamber. Similarly solid paraffin is melted in thereservoir of the second module (second reservoir 4) from which the waxsolution is typically transferred to the vacuum retort 3 at the start ofdaily operation and returned to the second reservoir 4 at the end of theday. The temperature of the solution is maintained at about 62° C. inthe microwave retort 2 and at about 65° C. in the vacuum retort 3.Tubing between (i) first reservoir 1 and microwave retort 2 or (ii)second reservoir 4 and vacuum retort 3 provide two-way fluidcommunication. The two reservoirs 1 and 4 for non-aqueous and waxsolutions are separate. The presence or absence of solution and/orbasket in a retort 2 or 3 may be determined with a level sensor todetect the volume or displacement of the solution.

A perforated basket containing tissue specimens in cassettes is loaded.A loading station is optional so the basket may be placed in a reactionchamber of the loading station (if present) which contains non-aqueousadmixture, on a conveyance which transfers a basket to the microwaveretort 2 (and subsequently to the vacuum retort 3), or directly into thereaction chamber of the microwave retort 2. The conveyance is preferablya robot arm with a hook which reversibly attaches to the basket; a panwith a replaceable absorbent liner is attached to the robot arm andswivels under the basket to catch dripping non-aqueous admixture or waxsolution. A lid is attached to each reaction chamber by a hinge; eachlid forms a seal with the reaction chamber using a rubber gasket, and isopened/closed by a chain driven with an electric motor M. Finally, whentissue impregnation is complete, the loaded basket is transferred fromthe vacuum retort 3. An unloading station is optional and a reactionchamber of the unloading station (if present) is empty so that excessmolten paraffin can drain from the basket. The time required to transferthe basket between stations is less than about 10 seconds. The tissuecassettes can then be unloaded from the basket.

The process described in Example 2 or 3 may be used in this system. Anon-aqueous admixture is transferred between microwave retort 2 andheated reservoir 1. The basket is transferred into an empty reactionchamber of microwave retort 2, the lid of the reaction chamber isclosed, non-aqueous admixture is transferred into the microwave retort 2from the heated first reservoir 1 to contact tissue specimens,non-aqueous admixture is returned to the heated first reservoir 1 whenincubation is complete (i.e., tissue specimens are suitably hardened),the lid is opened and the basket is transferred to the vacuum retort 3containing wax solution, and tissue specimens are incubated thereinuntil impregnation is completed.

The reaction chamber containing an impregnation agent is heated using aradiant heating source. Alternatively, a heater maintains thetemperature of water circulating in tubing in contact with theimpregnation agent to keep it in a molten state. For example, a coil oftubing can be located inside the reaction chamber; this heating coilwould then transfer heat to the contents. Preferably the heating coil iseliminated by wrapping the outside wall of the reaction chamber withelectrical wire that conducts heat through the walls into the contentsof the reaction chamber. Agitation in the vacuum retort can be performedby P/V cycles of nominal pressure 0.35 Kg/cm² and 500 mm Hg vacuum.

Other conditions (e.g., times and temperatures of each incubation) areas described in Examples 2-3. The system may be enclosed in a cabinet tocontain any fumes which may be present and to vent them (fume control).A tabletop system may use a mechanical conveyance to transfer tissuespecimens or they may be manually transferred. Alternatively, a doorwith a glass window at torso height allows the operator to access thesystem when there is no movement of the basket, to load a basket in orto unload a basket from the system, and to observe movement of thebasket. For safety, it is preferred that the door locks when the arm orlid on a retort is moving. Another door at knee height allows theoperator (i) to attach a bottle of the non-aqueous admixture to a portleading to the first reservoir 1 which is connected to the microwaveretort 2 and (ii) to melt paraffin in the second reservoir 4 which isconnected to the vacuum retort 3.

In an alternative embodiment (FIG. 1B), a single retort 5 may be usedwith both microwave and vacuum functions combined in the same location.A non-aqueous admixture from a first reservoir 6 and a wax solution froma second reservoir 7 are successively transferred into themicrowave/vacuum retort 5 to eliminate having to transfer tissuespecimens between two retorts.

Example 5 Detection of Antigen in Tissue Sections

Tissue specimens were fixed in formalin or UM-FIX, and then processedfor 30 minutes in each of the two reactions. Paraffin sections were cuton a microtome to a thickness of 3 microns, placed in a water bath, andfloated onto a glass slide. Paraffin was melted by placing slides ineither a 58° C. oven for 30 minutes, or preferably in a 37° C. oven forapproximately 18 hours or overnight, and then dewaxed in a xylene bathfor 10 minutes. Slides were rehydrated in decreasing ethanol solutionsfor one minute each (two baths of absolute alcohol, two baths of 95%alcohol, and one bath of 90% alcohol), and then rinsed by submerging intap water for 2 minutes.

Endogenous peroxidase was blocked with a solution of 6% hydrogenperoxide (H₂O₂) and methanol, or 35 ml of 6% H₂O₂ with 140 ml ofmethanol, incubated for 15 minutes. Slides were rinsed by submerging intap water for 2 minutes and phosphate buffered saline (PBS) for 2minutes, then dried.

Slides were transferred to a humidity chamber and normal horse serum(NHS) was added to block for 10 minutes. Excess normal horse serum wasdecanted from slides, and specific primary antibody was incubated for 30minutes on the tissue section in a humidity chamber at room temperature.Slides were flushed with PBS with a back-and-forth motion using asqueeze bottle, submerged in a PBS bath for 2 minutes, and excess PBSwas dried off each slide. Linking solution (also known as secondaryantibody or biotinylated anti-rabbit or anti-mouse) was added to eachtissue section and incubated for 25 minutes in a humidity chamber atroom temperature. Such rabbit, rat, and mouse secondary antibodies(e.g., anti-IgM, anti-IgG) may be obtained from Dako (Carpinteria,Calif.) and used at a dilution of about 1:600. Slides were flushed withPBS using a squeeze bottle, submerged in a PBS bath for 2 minutes, andexcess PBS is dried off each slide.

Signal was developed according to the manufacturer's instructions(Vector Laboratories). Avidin-biotin complex (ABC) solution was added tothe tissue section and incubated for 25 minutes in humidity chamber.Slides were flushed with PBS in a squeeze bottle and submerged in a rackin a PBS bath for 2 minutes. The rack was submerged in a bath ofdiaminobenzidine (DAB) chromogen for 6 minutes, then submerged underrunning water to wash gently for 4 minutes. Tissue sections werecounterstained with hematoxylin (staining time will depend on the age ofthe hematoxylin) from about 15 seconds to 90 seconds at roomtemperature. Slides were washed under running water for 3 minutes toremove excess counterstain, dehydrated in alcohol baths (about 10seconds in each) from 85% alcohol to absolute alcohol, cleaned inxylene, and coverslipped. Results are shown in Table 2.

TABLE 2 Antibody staining of tissue sections (m,. monoclonal; p,polyclonal) Tissue Fixative Antibody Staining Comments Prostate Formalincytokeratin HMK + staining confined to benign (m M630, Dako) glands,focus of adenocar- cinoma is negative AMACR (p 5045, Dako) − TonsilFormalin CD20 (m M755, Dako) + pattern of reactivity as expected CD10 (mNCL-DC10- + 270 Vector) CD3 (p A0452, Dako) + Liver Formalin HepPar 1 +staining confined to (m M7158, Dako) hepatocytes cytokeratin 7 +selective staining of (m M7018, Dako) bile duct epithelium Lung UM-FIXthyroid transcription +/− positive in pneumo- factor-1 cytes; negative(m M3575, Dako) in adenocarcinoma P63 (m P09-OP09L, +/− positive inbasal Oncogene Science) bronchial epithelium; tumor negative BreastFormalin CD31 (m M823, Dako) + endothelium Kidney Formalin renal cellantigen + (m NCL-RCC, Vector) glomerular epithelial + protein (mBioGenex) Lung UM-FIX cytokeratin cocktail + no tumor, strongly positive(m M3515, Dako) in pneumocytes and bronchial epithelium CD68 (m M814,Dako) + histiocytes Spleen Formalin FVIII-related antigen + (m M616,Dako) CD45 (m M701, Dako) + Placenta UM-FIX human chorionic gona- +dotropin (p A231, Dako) human placental lectin + (p A137, Dako) P57 (mMS1062-P, + Lab Vision) CD34 (m 347660 B-D) + Myeloperoxidase + (p A324,Dako) Small bowel Formalin chromogranin A + (m M869, Dako) REToncoprotein + (m NCL-RET, Vector) Appendix Formalin serotonin + (m M758,Dako) Breast Formalin estrogen receptor + fibrocystic disease- (1D5) (mM7047, Dako) weak reaction progesterone receptor + strongly positive (mM3569, Dako) Kidney UM-FIX P63 (m M7247, Dako) + transitional cellcarcinoma cytokeratin 20 − (m M7019, Dako) Skin UM-FIX epithelialmembrane + sebaceous glands antigen (m M613, Dako) T-F antigen +sebaceous glands (m M0898, Dako) Liver Formalin CD68 (m M814, Dako) +Kupffer cells cytokeratin 7 + bile duct epithelium (m M7018, Dako)Breast Formalin S100 protein + myoepithelial cells (p Z311, Dako) UterusFormalin desmin (m M760, Dako) + myometrium and vessel wall progesteronereceptor + myometrium (m M3569, Dako)

Example 5 DNA Extraction from Processed Tissue Sections

Two six micron tissue sections are placed in a 1.5 ml microfuge tube,800 μl xylene is added and mixed by vortexing, 400 μl absolute ethanolis added and mixed by vortexing, the tube is centrifuged for 5 minutesin a microfuge, and the supernatant is decanted. To the pellet, 800 μlabsolute ethanol is added and mixed by vortexing.

The supernatant is decanted after centrifugation, then 100 μl of adetergent and proteinase K solution (1% NP40 or Triton X-100, 2.4 μl of2.5 mg/ml proteinase K) is added to the pellet and incubated at 55° C.for one hour. Proteinase K is inactivated by incubation at 95° C. for 10minutes. The supernatant containing DNA is collected aftercentrifugation in a microfuge for 5 minutes. This material is ready forPCR. It should be precipitated and/or extracted further if Southernblotting is planned. More sections would be required to obtain enoughDNA for restriction analysis.

Example 6 RNA Extraction from Processed Tissue Sections

Ten sections (7 μm each) from a block of processed tissue were choppedusing disposable blades. They were placed in 50 ml Falcon tubes,deparaffinized with 20 ml of xylene, and the remaining tissue was thenwashed twice with absolute alcohol for 30 minutes. The tissue wassuspended at 0.5 gm/ml in a solution containing 4M guanidiniumthiocyanate, 25 mM Na citrate pH 7.0, 0.5% N-laurylsarcosine, and 0.1 Mof 2-mercaptoethanol. The solution was mixed by vortexing and DNA wassheared by passage through an 18 to 22 gauge syringe needle.

The RNA-containing solution was carefully layered on 2.8 ml of 5.7 MCsCl in several 5 ml centrifuge tubes (Sorvall), and RNA was sedimentedby centrifugation in an SW55Ti rotor at 35,000 rpm and 18° C. for 14hours in a Beckman L8-53 ultracentrifuge. The top fraction was carefullyremoved to leave an RNA pellet at the bottom of the tube. The pellet wasresuspended with ribonuclease-free water, and the Eppendorf tube wasspun at 14,000 rpm for 10 minutes. The supernatant containing RNA wassaved and the ultraviolet (UV) absorbance was measured: an extinctioncoefficient of 1 OD₂₈₀/cm is estimated to be the equivalent of about 40μg/ml RNA and the OD₂₆₀/OD₂₈₀ ratio should be between about 1.8 andabout 2.0.

Pieces of liver from two mice were placed in UM-FIX for approximatelytwo hours and then processed in accordance with Example 3. Tissue wasprocessed for either 15 minutes or 30 minutes in each of two reactions.The 18S and 28S rRNA bands of were separated by denaturing gelelectrophoresis (FIG. 2). RNA extracted from fresh mouse liver was usedas the positive control (lane C). RNA was extracted from processed livertissue of two mice (lanes 1 and 2 from the first mouse; lanes 3 and 4from the second mouse). Lanes 1 and 3 were processed for 15 minutes ineach reaction; lanes 2 and 4 were processed for 30 minutes in eachreaction. The 18S and 28S rRNA bands were present in the expected ratioand degradation was not seen.

In stating a numerical range, it should be understood that all valueswithin the range are also described (e.g., one to ten also includesevery integer value between one and ten as well as all intermediateranges such as two to ten, one to five, and three to eight). The term“about” may refer to the statistical uncertainty associated with ameasurement or the variability in a numerical quantity which a personskilled in the art would understand does not affect operation of theinvention or its patentability.

All modifications and substitutions that come within the meaning of theclaims and the range of their legal equivalents are to be embracedwithin their scope. A claim using the transition “comprising” allows theinclusion of other elements to be within the scope of the claim; theinvention is also described by such claims using the transitional phrase“consisting essentially of” (i.e., allowing the inclusion of otherelements to be within the scope of the claim if they do not materiallyaffect operation of the invention) and the transition “consisting”(i.e., allowing only the elements listed in the claim other thanimpurities or inconsequential activities which are ordinarily associatedwith the invention) instead of the “comprising” term. Any of these threetransitions can be used to claim the invention.

It should be understood that an element described in this specificationshould not be construed as a limitation of the claimed invention unlessit is explicitly recited in the claims. Thus, the granted claims are thebasis for determining the scope of legal protection instead of alimitation from the specification which is read into the claims. Incontradistinction, the prior art is explicitly excluded from theinvention to the extent of specific embodiments that would anticipatethe claimed invention or destroy novelty.

Moreover, no particular relationship between or among limitations of aclaim is intended unless such relationship is explicitly recited in theclaim (e.g., the arrangement of components in a product claim or orderof steps in a method claim is not a limitation of the claim unlessexplicitly stated to be so). All possible combinations and permutationsof individual elements disclosed herein are considered to be aspects ofthe invention. Similarly, generalizations of the invention's descriptionare considered to be part of the invention.

From the foregoing, it would be apparent to a person of skill in thisart that the invention can be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments should be considered only as illustrative, not restrictive,because the scope of the legal protection provided for the inventionwill be indicated by the appended claims rather than by thisspecification.

1-34. (canceled)
 35. A method for tissue processing of one or moretissue specimens, wherein the method uses only two different chemicalcompositions, comprising: (a) initially impregnating a tissue specimenwith a single non-aqueous admixture comprised of (i) fixing, (ii)dehydrating, and (iii) clearing functions which is heated; and then (b)substantially impregnating the tissue specimen with a single waxsolution which is heated and also under less than atmospheric pressure.36. The tissue processing method of claim 35, wherein the tissuespecimen is about 3 mm or less in thickness.
 37. The tissue processingmethod of claim 35, wherein tissue specimens are manually processed. 38.The tissue processing method of claim 35, wherein tissue specimens aremechanically processed.
 39. The tissue processing method of claim 35,wherein the tissue specimen is processed without using an aldehyde. 40.The tissue processing method of claim 35, wherein the tissue specimen isprocessed without using a xylene.
 41. The tissue processing method ofclaim 35, wherein the tissue specimen has not been fixed prior toprocessing.
 42. The tissue processing method of claim 35, wherein thetissue specimen has been fixed in formaldehyde prior to processing. 43.The tissue processing method of claim 35, wherein the tissue specimenhas been preserved in methanol and polyethylene glycol (PEG) prior toprocessing.
 44. The tissue processing method of claim 35, wherein thetissue specimen is processed in a microwave retort for (a).
 45. Thetissue processing method of claim 44, wherein the tissue specimen issubstantially hardened by the non-aqueous admixture, radiation from themicrowave unit, or both.
 46. The tissue processing method of claim 35further comprising agitation to promote chemical exchange between thenon-aqueous admixture and the tissue specimen.
 47. The tissue processingmethod of claim 35, wherein the non-aqueous admixture is above about 55°C. and/or below about 75° C.
 48. The tissue processing method of claim35, wherein the non-aqueous admixture is comprised of fixative anddehydrant in a volume ratio greater than about 1:6 and/or less thanabout 6:1.
 49. The tissue processing method of claim 35, wherein afixative is greater than about 20% (v/v) and/or less than about 60%(v/v) of the non-aqueous admixture.
 50. The tissue processing method ofclaim 35, wherein a dehydrant is greater than about 20% (v/v) and/orless than about 60% (v/v) of the non-aqueous admixture.
 51. The tissueprocessing method of claim 35, wherein a clearant is greater than about10% (v/v) and/or less than about 30% (v/v) of the non-aqueous admixture.52. The tissue processing method of claim 35, wherein the non-aqueousadmixture is further comprised of a surfactant.
 53. The tissueprocessing method of claim 35, wherein the non-aqueous admixture iscomprised of a fixative which is at least one ketone and a dehydrantwhich is at least one alcohol.
 54. The tissue processing method of claim35, wherein the non-aqueous admixture is comprised of a clearant whichis mineral oil.
 55. The tissue processing method of claim 35, whereinthe non-aqueous admixture is further comprised of dimethyl sulfoxide(DMSO).
 56. The tissue processing method of claim 35, wherein thenon-aqueous admixture is comprised of about 15% (v/v) to about 35% (v/v)acetone, about 45% (v/v) to about 65% (v/v) isopropyl alcohol, and about10% (v/v) to about 25% (v/v) mineral oil.
 57. The tissue processingmethod of claim 35, wherein the non-aqueous admixture is comprised ofabout 40% to about 60% (v/v) of acetone, about 25% to about 35% (v/v) ofisopropanol, and about 10% (v/v) to about 25% (v/v) of mineral oil. 58.The tissue processing method of claim 35, wherein the tissue specimen issubstantially hardened after being incubated in the non-aqueousadmixture for about 15 minutes or less.
 59. The tissue processing methodof claim 35, wherein the tissue specimen is substantially hardened afterbeing incubated in the non-aqueous admixture for about 30 minutes orless.
 60. The tissue processing method of claim 35, wherein the tissuespecimen is substantially hardened after being incubated in thenon-aqueous admixture for about 45 minutes or less.
 61. The tissueprocessing method of claim 35, wherein the tissue specimen is processedin a vacuum retort for (b).
 62. The tissue processing method of claim61, wherein the tissue specimen is substantially impregnated at apressure above about 100 torr and/or below about 760 torr.
 63. Thetissue processing method of claim 35, wherein the wax solution is aboveabout 55° C. and/or below about 85° C.
 64. The tissue processing methodof claim 35, wherein the wax solution has been degassed and dehydrated.65. The tissue processing method of claim 35, wherein the wax solutionis made from paraffin which is solid below about 30° C.
 66. The tissueprocessing method of claim 35, wherein the tissue specimen can besectioned after being incubated in the wax solution for about 15 minutesor less, and then cooled.
 67. The tissue processing method of claim 35,wherein the tissue specimen can be sectioned after being incubated inthe wax solution for about 30 minutes or less, and then cooled.
 68. Thetissue processing method of claim 35, wherein the tissue specimen can besectioned after being incubated in the wax solution for about 45 minutesor less, and then cooled.
 69. A method for tissue processing of one ormore tissue specimens using only two different chemical compositions andan apparatus comprising: a first module comprising (i) a first reactionchamber with an interior geometry comprised of a whispering gallery toachieve uniform distribution of microwave energy and heating of theinterior, (ii) a single non-aqueous admixture which is optionallycontained within the first reaction chamber, (iii) a source of microwaveenergy which is transmitted into the first reaction chamber to heat thenon-aqueous admixture, and (iv) an agitator within the first reactionchamber to promote chemical exchange between the non-aqueous admixtureand the tissue specimen and a second module comprising (i) a secondreaction chamber, (ii) a single wax solution which is optionallycontained within the second reaction chamber, (iii) a source of radiantenergy which is transmitted into the second reaction chamber to heat thewax solution, and (iv) a vacuum pump connected to the second reactionchamber; the method comprising: (a) initially impregnating a tissuespecimen in the first module with the single non-aqueous admixturecomprised of (i) at least one ketone, (ii) at least one alcohol, and(iii) mineral oil while being agitated and heated then (b) substantiallyimpregnating the tissue specimen in the second module with the singlewax solution while being under vacuum and heated.